Protective Effects Of Taurine On The Alterations Of Retinal Müller Cells In Short-term Diabetic Rats And Its Mechanism

Diabetes mellitus is increasing worldwide at an alarming rate. Diabetic retinopathy (DR) is a common complication of diabetes and a leading cause of legal blindness in working-age adults. The clinical hallmarks of DR include increased vascular permeability, leading to edema, and endothelial cell proliferation. The mainstay of therapy for DR is panretinal photocoagulation. Other agents that are known to decrease the risk of developing DR include controlling blood glucose levels and blood pressure, however, some patients develop retinopathy despite good control of blood glucose and blood pressure. Thus new therapies able to prevent the development or progression of DR are needed. Much of the research effort has been focused on vascular changes, but it is becoming apparent that other degenerative changes occur beyond the vascular cells of the retina. These include the altered expression of glial fibrillary acidic protein (GFAP) and vascular endothelial growth factor (VEGF) in the Müller cells, and always combined with alteration in glutamate metabolism.Müller cells, the main glial cell within the retina, are vital for maintaining the normal health of the retina and have been implicated in many retinal diseases, including DR. Recently, some studies have demonstrated that the increased Müller cells'reactivity and Müller cells'glutamate dysmetabolism are early pathogenic events, which always resulted in an increment in retinal glutamate levels in diabetes. Increased glutamate can accelerate the death of retinal vascular cells and nonvascular cells, which might play an important role in the pathogenesis of retinopathy. A major physiological function of Müller cells is to regulate the ionic and molecular composition of the retinal microenvironment. An intensively investigated function of Müller cells was assumed as their ability to eliminate synaptically released glutamate, a neurotransmitter with more than 90% of the synapses placed in the retina. Glutamate transporters (GLAST), glutamine synthetase (GS) and glutamate decarboxylase (GAD) were found in Müller cells. So, it was postulated that synaptically released glutamate in retina was absorbed by Müller cells via GLAST transportation and rapidly converted to glutamine andγ-aminobutyric acid (GABA) with the aid of GS and GAD. The resulting glutamine was then returned to the neurons for glutamate re-synthesis. By this way, the neurotransmitter pool was replenished and glutamate neurotoxicity has been successfully prevented.Taurine (2-aminoethanesulfonic acid), a sulphur-containingβ-amino acid, is one of the most abundant free amino acids in various tissues, and has been proved to play important roles in numerous physiological functions, such as osmoregulation, modulation of neurotransmitters, membrance stabilization, and antioxidation in mammals. It was concluded that intracellular depletion of taurine contributes much to chronic cytotoxicity in diabetes. Several studies have recently indicated that taurine exhibited beneficial effects in adults with DM. A lower level of taurine was clinically obtained in insulin-dependent and insulin-independent DM patients, taurine was decreased in plasma and platelets. In addition, an inverse correlation between the log of plasma taurine and glycosylated hemoglobin has been found. Moreover, in insulin-dependent diabetic patients, taurine supplementation decreased platelet aggregability, restored its own plasma and platelet levels, and abolished the inverse correlation between log plasma taurine and glycosylated hemoglobin. Moreover, the long-term dietary taurine supplementation retarded the morbidity of diabetes.Based on the analysis above, we presume taurine maybe an effective protective agent of Müller cells changes induced by diabetes. The aim of this study was to investigate the effects of taurine supplementation on diabetes-induced Müller cells alterations in rats retina and the associated molecular mechanism. The Sprague-Dawley (SD) rats model of streptozotocin (STZ)-induced diabetes was used in vivo study. In vitro study, Müller cells cultured in high glucose were used as the model. TUNEL, immunohistochemistry, immuocytochemistry, amino acid analysis, laser confocal analytical assay, flow cytometry, RT-PCR and western blotting method were used to study the structural and functional changes induced by diabetes and high glucose. Meanwhile the proective effects of taurine and the associated molecular mechanism of taurine transporter, glutmate clearance pathway were deeply studied. The main results and conclusions were summarized as follows:1. The inner layer cells arranged irregularly and the intercellular spaces became wide in diabetic rats retina. The cells'number in inner nuclear layer (INL) and ganglion cell layer (GCL) was decreased obviously. Increased electron density, chromatin margination and karyopycnosis/karyorrhexis were observed easily in retinal Müller cells of diabetic rats. Some vacuolation appeared in the cytoplasm of Müller cells. Taurine treatment could significantly improve the changes of histopathology and ultrastructure in diabetic rats retina. Although no significant change has been observed with blood glucose content in taurine treatment group, taurine could protect the structural damages of retina induced by diabetes effectively, which indicating that the protective mechanisms of taurine was not by decreasing the levels of blood glucose in diabetic rats.2. The implicit times of a and b wave of rod-response, b wave of max-response, p4 wave of OPs and b wave of phot-ERG of diabetic rats were respectively prolonged significantly compared to those of control ones. The Os1 amplitude of OPs was significantly decreased in diabetic rats. Although no significant improvement in 1% taurine treatment group, 5% taurine treatment could significantly reverse the changes of above wave, which suggesed that the functions of Müller cells changed in the early stage of diabetic rats and could be protected by taurine treatment.3. In vivo study, some TUNEL-positive cells were observed in diabetic rats retina at 2 weeks. In 12 weeks, there were more TUNEL-positive cells found in diabetic rats retina than that in normal controls and the TUNEL-positive cells were 33% in total cells. When treatment with taurine, the TUNEL-positive percent was markedly decreased. In vitro study, increased karyopycnosis/karyorrhexis and some apoptotic bodies were found in high glucose cultured Müller cells. rate of apoptosis in Müller cells incubated in high glucose increased significantly (36.52% vs 0.34% in controls). Treatment with taurine could inhibit apoptosis (the apoptosis rate was 16.43%, 3.75% and 2.01% in low-dost, meta-dose and high-dose taurine group respectively).4. A common marker for reactive gliosis is the well-described increase in the expression of GFAP. VEGF is a potent angiogenic growth factor. RT-PCR and western-blotting analysis showed increases in GFAP, VEGF mRNA and protein expression in STZ-induced diabetic retina and high glucose cultured Müller cells compared to normal controls. Treatment with taurine significantly blocked the increases in GFAP, VEGF mRNA and protein expression in diabetic retina and high glucose cultured Müller cells.5. The level of taurine was decreased in untreated diabetic rats and was repleted by taurine supplementation. Our study suggested that the expression of TauT was reduced in diabetes and high glucose group and could be stimulated expression by taurine supplementation.6. The concentration of glutamate was increased in untreated diabetic rats vs nondiabetic controls, and this increase was prevented by taurine. the expressions of GLAST, GS and GAD were reduced in diabetes and high glucose group and could be stimulated expression by taurine supplementation. The ability of L-[2,3-3H]-glutamate uptake was attenuated in Müller cells in high glutamate cultured group and could be potentized by taurine.In brief, Our studies revealed that taurine ameliorates diabetic retinopathy possibly via increasing the ability of taurine transporter and relieving anti-excitotoxicity of glutamate, which suggest a possible utility of taurine as an adjuvant therapeutic agent in the management of diabetes and its complications.